International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-5, Sept-Oct- 2018

http://dx.doi.org/10.22161/ijeab/3.5.21 ISSN: 2456-1878

Tulsi (Ocimum sanctum), excellent source of

phytochemicals
Borah R1; Biswas S. P.2
Department of Life Sciences, Dibrugarh University, Assam, India
Email: [email protected] m
[email protected]

Abstract— Ocimum sanctum also known as Tulsi or they are rich in a wide variety of secondary metabolites
Holybasil is an aromatic plant and it belongs to the such as tannins, phenolics, alkaloids and flavonoids etc
family Lamiaceae. It is widely used as medicine to cure which enhances growth, innate immune response and
various ailments. The objective of the study was to disease resistance against pathogenic bacteria in human as
analyse different phytochemical components of tulsi leaf. well as in different organisms (Edoga et al., 2005). About
The dried powder of Tulsi (50g) was placed in the thimble 80% of individuals from developed countries use various
of Soxhlet apparatus and the experiment was done medicinal plants as traditional medicines as anticancer
separately for methanol, ethanol and distilled water. The drugs (Dewick, 1996), antimicrobial drugs (Phillipson,
percentage yield was 8%w/w,7%w/w, and 5%w/w 1996), antifungal and in various proposes. The medicinal
respectively. The study reveals that various secondary plants are rich sources of secondary metabolites which are
metabolites such as carbohydrate, tannin, flavonoids, chemically and taxonomically extremely diverse
saponins, glycoside, terpenoid, fatty acids and phenol are compounds with obscure function. A large number of
present in tulsi leaf extract. From the quantitative phytochemicals are widely uses in human therapy,
analysis it was found that high amount of phenols are agriculture, veterinary, various scientific researches and in
present in Tulsi leaf ranging from 1.6 to 7.6 percentages. different areas (Vasu et al., 2009) along with inhibitory
Consequently the amount of alkaloid and flavonoids effects on all types of microorganisms in vitro (Cowan,
ranged from 0.91 to 1.28 and 1.56 to 2.24 percentages 1999).
respectively. From the GC-MS analysis of methanolic Ocimum sanctum L. commonly known as holy basil
extract three compounds were identified as major (Tulsi) is an herbaceous perennial, belongs to family
constituents viz., Eugenol , Benzene, 1, 2-dimethoxy- 4- Lamiaecae and is considered as one of the most important
(2- propenyl), α - Farnesene and Cyclohexane, 1, 2, 4- source of medicine and drugs with many
triethenyl. Thesephyto-chemicals are known to possess secondarymetabolites and essential oils recommended
antiseptic, analgesic, anti-inflammatory, antimicrobial, fortreatment of malaria, diarrhoea, bronchial
antistress, immunomodulatory, hypoglycemic, hypotensive asthma,dysentery, bronchitis, skin diseases, arthritis,
and antioxidant properties. Hence it is more beneficial to painfuleye disease, chronic fever and eye diseases etc 5,6.
use tulsi asan herbal medicine as compare to chemically Inaddition, Ocimum sanctum also showsanticancerous,
synthesized drug. antifungal, antimicrobial, antifertility,hepatoprotective,
Keywords— Ocimum sanctum, phytochemical, antispasmodic, cardio protective,antiemetic, antidiabitic,
medicine, GC-MS. analgesic, adaptogenic, anddiaphoretic properties6-9.The
pharmacological studies reported in the present research
I. INTRODUCTION confirm the therapeutic value of O. Sanctum. Therefore,
The plant kingdom is an excellent source of potential the present study looks into the extraction and preliminary
drugs and in the recent years there has been an increasing phytochemical analysis of O. Sanctum leaves.
awareness about the importance of medicinal plants.
Medicinal plants are rich source of different types of II. MATERIALS AND METHODS
medicines and produce various bioactive molecules. Collection of plant material: Leaves of Ocimum
Herbal plant extracts are very useful and are the major sanctum L. (tulsi) were collected from different sites of
sources of medicine which play vital role in controlling Dibrugarh District, Assam, washed with sterile water and
various types of pathogens (Doss, 2009) and as growth dried in shades. Then the samples were powered in
promoters. These are the cheaper source for therapeutics mechanical grinder.
and viable solution for various pathogens.The medicinal Aqueous, methanol and ethanol extract: The dried tulsi
plants extract have now emerged as a good alternative as (50g) powder was placed in the thimble of Soxhlet

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-5, Sept-Oct- 2018
http://dx.doi.org/10.22161/ijeab/3.5.21 ISSN: 2456-1878
apparatus and 500- 700 ml of distilled water, methanol Test for tannin: 1 ml of distilled water and 2-3 drops of
and ethanol was used for extraction procedure and the ferric chloride solution was added to 0.5 ml of crude
experiment was done separately for all the two solvents extract. A black coluration indicated the presence of
and distilled water. The extraction was continued till clear tannin.
solvent or water was seen in the thimble. The extract was Test for flavonoi ds
concentrated using rotary evaporator. Then the extract Shinoda test: Crude extract was mixed with small amount
was dried in a digital water bath till a dark green residue of magnesium and concentrated HCl was added drop
was obtained. The percentage yield of the extract was wise. Appearance of pink scarlet colour after few minutes
calculated using the following formula: indicated the presence of flavonoids.
Final weight of the dried extract Alkaline reagent test: 0.5 ml of crude extract was mixed
Percentage yield = ----------------------------------------- × with 2ml of 2% solution of NaOH. An intense yellow
100 colour was formed which turned colourless on addition of
Initial weight of the powder few drops of diluted acid which indicated the presence of
All the three extracts were kept in separate vials in the flavonoids.
refrigerator till further use. Test for saponins: 1ml of crude extract was mixed with
Qualitative phytochemical analysis: The extract was 5ml of distilled water in a test tube and it was shaken
tested following standard biochemical methods as vigorously. The formation of stable foam was taken as an
described below. indication for the presence of saponins.
Test for proteins: Test for glycosides
Biuret’s test: 2ml of Biuret reagent was added to 2ml of Liebermann’s test: Crude extract was mixed with each of
extract. The mixture was shaken well and warm for 5 min. 2ml of chloroform and 2ml of acetic acid. The mixture
Appearance of red or violet colour indicated presence of was cooled in ice. Carefully concentrated H2 SO4 was
proteins. added. If colour change from violet to blue to green which
Million’s test: Crude extract was mixed with 2ml of indicated the presence of steroidal nucleus, i.e., glycone
Millon’s reagent, if precipitate appeared which turned red portion of glycoside.
on gentle heating confirmed the presence of protein. Salkowski’s test: 2ml of chloroform was mixed with crude
Ninhydrin test:Crude extract was mixed with 2 ml of extract. Then 2ml of concentrated H2 SO4 was added
0.2% solution of Ninhydrin and boiled for some time, if carefully and shaken gently. A reddish brown colour
violet colour appeared indicating the presence of amino indicated the presence of glycoside.
acids and proteins. Keller-kilani test:0.5 ml of crude extract was mixed with
Test for carbohydrates: 2ml of glacial acetic acid containing 2-3 drops of 2%
Fehling’s test: Equal amount of Fehling A and Fehling B solution of FeCl3. Then 2ml of concentrated H2 SO4 was
reagents were mixed and 2ml of it was added to the plant poured into the mixture. A brown ring at the interface
extract and then gently heated the sample. Appearance of indicated the presence of cardiac glycosides.
brick red precipitate indicated the presence of reducing Test for steroid
sugars. (i) 2ml of chloroform was added to the crude
Benedict’s test: Crude extract when mixed with 2ml of extract of Tulsi. Then 2ml of each of concentrated H2 SO4
Benedict’s reagent and boiled, a reddish brown precipitate and acetic acid were added into the mixture. The presence
formed which indicated the presence of the carbohydrates. of steroids was indicated by appearance of a greenish
Molisch’s test: 2ml of Molisch’s reagent was added to 0.5 coloration in the reaction mixture.
ml of crude extract and the mixture was shaken properly. (ii) Crude extract was mixed with 2ml of chloroform and
After that, 2ml of concentrated H2 SO4 was poured gently added concentrated H2 SO4 . A red colour was seen
carefully along the side of the test tube. Appearance of a in the lower layer this indicated the presence of steroids.
violet ring at the interface indicated the presence of Test for terpenoids: Crude extract was mixed in 2ml of
carbohydrate. chloroform and evaporated to dryness. To this, 2ml of
Iodine test: 2ml of iodine solution was mixed with 0.5 to concentrated H2 SO4 was added and heated for about 2
1 ml of crude extract. A dark blue or purple coloration minutes. Presence of terpenoids was indicated by a
indicated the presence of the carbohydrate. greyish colour at the interface.
Test for phenol: 2 ml of alcohol and 2-3 drops of ferric Test for alkaloids: 2ml of 1% HCl was mixed with crude
chloride solution was added to 1 ml of crude extract, blue- extract and heated gently. After heating, Mayer’s And
green or black coloration indicated the presence of Wagner’s reagents were added to the mixture. If
phenols precipitate was observed in the reaction mixture which
indicated the presence of alkaloids.

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-5, Sept-Oct- 2018
http://dx.doi.org/10.22161/ijeab/3.5.21 ISSN: 2456-1878
Test for anthraquinone: 5ml of chloroform and 5 ml of Methanolic extract of the leaves of Ocimum
ammonia solution was added to 0.2 gm of plant extract. sanctumwas subjected to GC-MS analysison a GC- MS
Appearance of pink, red or violet colour indicated the Clarus 500 Perkin Elmer systemcomprising a AOC- 20i
presence of anthraquinone. autosampler and gaschromatograph interfaced to a mass
Oils & Fats: A small quantity of crude extract was spectrometer (GC-MS) instrument employing the
pressed between two filter papers separately. An oily followingconditions: Restek RtxR – 5, (30 meter X 0.25
appearance on filter paper indicated the presence of fixed mm)(5% diphenyl / 95% dimethyl polysiloxane),
oil and fats. runningin electron impact mode at 70 eV; helium
Test for lactones (99.999%) was used as carrier gas at a constant flow
Baljet’s test:Crude extract was treated with sodium of1ml/min and an injection volume of 2.0 μl
picrate solution. Presence of lactone was observed by wasemployed(split ratio of 10:1); injector
appearance of yellow to orange colour in the mixture. 0
temperature280 C. The oven temperature was
Quantitative analysis of phytochemical in the plant programmedfrom 50°C (for 1 min.), with an increaseof 6
extract: 0 C / min to 280 0 C, then ending with aisothermal for

Determination of total phenolic contents (Singleton et 15min at 280°C. Mass spectra weretaken at 70 eV; a 0.5
al., 1999): The amount of total phenol for aqueous, seconds of scan interval andfragments from 40 to 550 Da.
methanol and ethanol extract were determined by Folin - Total GC runningtime was 60 minutes.
Ciocalteu reagent method. 2.5 ml of 10% Folin- Ciocalteu Identification of Compounds
reagentand 2 ml of 2% Na 2 Co 3 were added to 0.5 ml of Interpretation on mass spectrum GC-MS was conducted
plant extract. The mixture was then incubated at room using the database of department of Chemistry; Dibrugarh
temperature for 30 minutes. Gallic acid was used as University. The mass spectrum of the unknown
standard (1mg/ml). The absorbance of the sample was component was compared with the spectrum of the known
measured at 765nm. All the tests were done in triplicates components stored in the department of Chemistry
and the results were determined from standard curve and library.
were expressed as gallic acid equivalent (mg/g of
extracted compound). III. RESULTS
Determination of alkaloid (Harborne, 1973): 5 g of the The yield of residue after Soxhlet extraction and
sample was taken and 200 ml of 10% acetic acid in evaporation of 50 gm dried plant leaves in methanol,
ethanol was added to the sample and allowed to stand for ethanol and water were as follows:
4 hours. Then the solution was filtered and the extract was Table.1: Amount of plant extracts yield percentage in
concentrated on water bath Conc. NH4 (OH) was added different solvents
drop wise and the whole solution was allowed to settle Extract Yield amount (% )
and the precipitate was then washed with dilute W/W
ammonium hydroxide and filtered. The residue was dried Aqueous 5%
and weighed and this was the amount of alkaloid present Methanol 8%
in the plant material Ethanol 7%
Determination of flavonoids (Bohm & Kocipai-
Abyazan, 1994): 10 g of plant sample was taken and The phytochemicals analysis in Ocimum sanctum (Tulsi)
extracted repeatedly with 100ml 80% methanol. Then the leave extracts in the two solvents and aqueous conditions
solution was filtered and the filtrate was transferred into were summarized in Table 2. Various bioactive molecules
an empty crucible and evaporated into dryness over water were found in Tulsi leaf extract from the phytochemical
bath and weighed. The final weight dry weight was screening. The amount of extraction is more in case of
amount of flavonoids in the plant sample. organic solvent then that of water. From the quantitative
Preparation of stock solution analysis it was found that high amount of phenols are
The extracts were reconstituted in methanol. Methanolic present in Tulsi leaf ranging from 1.6 to 7.6 percentages.
extracts (1 μl) were injected for GC-MS analysis. Consequently the amount of alkaloid and flavonoids
Gas Chromatography-Mass Spectrometry analysis ranged from 0.91 to 1.28 and 1.56 to 2.24 percentages
respectively.

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-5, Sept-Oct- 2018
http://dx.doi.org/10.22161/ijeab/3.5.21 ISSN: 2456-1878
Table.2: Qualitative phytochemical screening methanol extract of tulsi leaf
Phytochemicals Aqueous Methanol Ethanol extract
extract extract

Protein - - -
Carbohydrate - + +
Phenol + + -
Tannin - + +
Flavonoid + + +
Saponin - + +
Glycosides + + +
Steroid - - -
Terpenoid - + +
Alkaloid + + +
Anthraquinone - - -
Fixed oils and fatty acid - + -
Test for lactones - - -
“+”present, “-” absent

Table.3: Percentage of total phenolic, alkaloid and flavonoid contents in plant extract
Extract Phenolic Alkaloid Flavanoi d
Aqueous 1.61±0.56 0.91±0.66 1.56±0.64
Methanol 7.61±0.55 1.28±0.03 2.24±1.02

Ethanol 4.61±0.56 0.94±0.58 1.91±0.56

Each value is the average of three analysis and ± standard deviation.

Table.4: Chemical constituents and the activity of some of the phytocomponents of Ocimum sanctum
Sl. Retention time Name of the Molecular Molecular Activity**
No (unit?) compounds weight formula
1. 7.20 Eugenol 164 C10 H12 O2 Anti- inflammatory,
antioxidant, anticancer, Acaricide,
Antibacterial,Antispasmodic,
Antiviral, Insecticide
2. 7.70 α - Farnesene 93 C15 H24 Acaricide, allergenic, analgesic,
anaesthetic, antibacterial, anti-
inflammatory, antiedemic, antioxidant,
antiviral, antitumor, antiulcer
3. 7.50 Benzene, 1, 2- 178 C11 H14 O2 Insect-attractant,
dimethoxy- perfumery, flavour antibacterial,
4-(1-propenyl) nematicide

4. 13.36 Cyclohexane,1,2,4- 162 C12 H18 Antibacterial, anti-inflammatory,

triethenyl antiedemic, antispasmodic
**Source: Dr. Duke’s phytochemical and ethnobotanical database (online database)

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-5, Sept-Oct- 2018
http://dx.doi.org/10.22161/ijeab/3.5.21 ISSN: 2456-1878

Fig.1: GC- MS chromatogram of the methanolic extract of the leaves of Ocimum sanctum

IV. DISCUSSION properties (Asquith et al., 1986). Flavanoids are

Ocimum sanctum has various properties such as antistress, responsible for antioxidant and immunostimulatory
antiseptic, analgesic, anti-inflammatory, antimicrobial, properties. According to Cragg et al., 1999 and Khanna et
immunomodulatory, hypoglycemic, hypotensive, al., 2003 alkaloids, glycosides, flavanoids and saponins
cardioprotective and antioxidant (Williamson, 2002, are antibiotic principles of plants and these antibiotic
Tanwar et al., 2015). Eugenol (1-hydroxy-2-methoxy-4- principles are actually the defensive mechanisms of the
allylbenzene), the active constituents present in O. plants against pathogens.
sanctum have been found to be largely responsible for the GC-MS chromatogram of the methanolic extract of
therapeutic potentials (Sailaja et al., 2010). This plant has Ocimum sanctum showed four major peaks (Figure.1) and
various properties such as antistress, antiseptic, analgesic, has been identified after comparison of the mass spectra
anti-inflammatory, antimicrobial, immunomodulatory, with the department of Chemistry library, DU, indicating
hypoglycemic, hypotensive, cardioprotective and the presence of four phytocomponents. It was observed
antioxidant (Williamson, 2002, Tanwar et al., 2015). that presence of Eugenol (Synonym: 2-Methoxy- 4-(2-
Eugenol (1-hydroxy-2-methoxy-4-allylbenzene), the propenyl) phenol), Benzene, 1, 2-dimethoxy- 4- (2-
active constituents present in O. sanctum have been found propenyl) - (synonym: Methyl- Isoeugenol), α - Farnesene
to be largely respons ible for the therapeutic potentials and Cyclohexane, 1, 2, 4- triethenyl were the major
(Sailaja et al., 2010). components in the extract. The phytochemicals that
The study reveals that various secondary metabolites such contribute to the medicinal property of the plant leaves is
as carbohydrate, tannin, flavonoids, saponins , glycoside, listed in Table No.1. Benzene, 1, 2-dimethoxy- 4-(1-
terpenoid, fatty acids and phenol are present in tulsi leaf propenyl) (Methyl-Isoeugenol) has the property of
extract. Leaves of Ocimum sanctum contain water-soluble Antifungal activity (Kurita et al., 1981), Nematicidal
phenolic compounds and various other constituents, such activity (Park et al., 2003) and Antifeedant activity
as eugenol, methyl eugenol and caryophylllene that may (Katsumi, 1987).Eugenol is reported to
act as an immunostimulant. Saponins act as anti- possessAntimycotic (Azzouz et al., 1982) Antiviral
hyperlipedemic, hypotensive and cardiodepessive (Bishop, 1995) Desinsection (Konstantopoulou et al.,
properties (Bairwaet al., 2012). The phytochemical 1992) Antiparasitic (Pandey et al., 2000) Antioxidant (Ou
constituents such as alkaloids, steroids, flavanoids, et al., 2006) Anticancer (Hussain et al., 2011) and Ant
tannins, phenols and several other aromatic compounds of insect activities(Pessoa et al., 2002).
plants serve a defense mechanism against predation by Leaves extract of O. sanctum affected both
many microorganisms, insects and other herbivore specific and non-specific immune responses and disease
(Bonjar et al., 2004) Glycosides can act as resistance against fungal and bacterial infection(Santra et
cardiostimulants in cases of cardiac failure (Sood et al., al., 2017). It stimulated both antibody response and
2005). Tannins have anti diarrheal and haemostasis neutrophil activity. The experimental studies have shown

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-3, Issue-5, Sept-Oct- 2018
http://dx.doi.org/10.22161/ijeab/3.5.21 ISSN: 2456-1878
that methanolic extract of Ocimum sanctum has [2] Azzouz, M.A. and Bullerman, L.B., 1982,
anticancer effect by inhibition of nitric oxide synthesis Comparative antimycotic effects of selected herbs,
(Kim et al., 1998). The use of medicinal plants acts as a spices, plant components and commercial anti fungal
source of antimicrobial agent also for aquaculture. In agents, Journal of Food Protection, 45(14): 1298-
Macrognathus pancalus, the extract of O. sanctum was 1301.
found to enhance the antibody response (Dugenci et al., [3] Bairwa, M.K, Jakhar1, J.K., Satyanarayana Y and
2003). The different leaf extracts of Tulsi (Ocimum Reddy, A.D. 2012, Animal and plant originated
sanctum), shows antimicrobial activity against three immunostimulant used in aquaculture , Scholars
human pathogens Escherichia coli, Staphylococcus Researchs Library, 2 (3):397-400.
aureus and Candida albicans. (Subramanian et al.,2014). [4] Bishop, C.D., 1995, Antiviral activity of the
Tulsi oil showed significant anti-inflammatory, analgesic, essential oil of Melaleuca alternifolia (Maiden and
antipyretic and antimicrobial effects. It has also shown Betche) Cheel (tea tree) against tobacco mosaic
memory enhancing, antifertility, anticataract, antithyroid, virus, Journal of Essential Oil Research,7(6): 641-
antiulcer, antidiabetic, antiarthritic, antiamnesic, 644.
antihelmenthic, anticataract, hepatoprotective and no [5] Boham, B.A. and Kocipai, A.R., 1994, Flavanoids
tropic activity (Rajesh et al., 2013). Alcoholic extract and condense tannins from leaves of Hawaiian
increased step down latency and acetyl cholinesterase Vaccinium vaticulatum and V. Calicinium. Pacific
inhibition and so used in the treatment of cognitive Science, 48: 458-463.
disorders. Ocimum sanctum has been widely employed in [6] Bonjar, GHS., Nik, AK. and Aghighi, S., 2004,
traditional medicines. Hence phytochemicals from this Journal of Biological Science, 4, 405-412.
plant can be used in variety of disorders afflicting [7] Cowan, M.M. 1999, Plant products as antimicrobial
mankind. The herbs are cheap, available in large quantity agents., Clinical Microbiological Review: 564-582.
around us and they pose no danger to the living [8] Dewick, P.M., 1996, Tumor inhibition from plants:
organisms, the environment and the consumers and hence Tease and Evans. Pharmacognosy.
greatly helpful for living organisms. [9] Doss, A., 2009, Preliminary phytochemical
screening of some Indian medicinal plants. Ancient
V. CONCLUSION Science of Life, 29(2): 12-16.
The presence of various bioactive compounds in the tulsi [10] Dugenci, S.K., Arda, N. and Candan, A., 2003,
leaves justifies the uses for various ailments by living Some medicinal plants as immunostimulant for fish.
population. The results confirm the use of Ocimum Journal of Ethnopharmacology, 88 (1): 99–106.
sanctum plant as traditional medicinal properties [11] Edoga, H.O., Okwu, D.E., Mbaebie, B.O. 2005,
andsuggest that some of the plant extracts possess Phytochemicals constituents of some Nigerian
compounds with antimicrobial properties that can be used medicinal plants. African Journal of Biotechnology,
asantimicrobial agents in new drugs for the therapy of 4(7): 685-688.
infectious diseases caused by various pathogens. It is [12] Harborne, J.B., 1973, Phytochemical methods.
more beneficial to use tulsi asan herbal medicine as London. Chapman and Hall, Ltd.
compare to chemically synthesized drug. [13] Hussain, A., Brahmbhatt, K. And Priyani, A., 2011,
Eugenol enhances the chemotherapeutic potential of
ACKNOWLEDGEMENT gemcitabine and induce anticarcinogenic and anti-
The authors acknowledge their profound gratitude to inflammatory activity in human cervical cancer
theDepartment of Life Sciences, Dibrugarh University for cells, Cancer Biother Radiopharm, 26(5): 519-27.
providing necessary facilities and encouraging us to carry [14] Katsumi, Y., 1987, Minor components from growing
out the work.The authors are also grateful to the buds of Artemisia capillaris that acts as insect
Department of Chemistry, Dibrugarh University antifeedants, Journal of Agricultural and Food
permitting for GC-MS analysis and also Thankful to Chemistry, 35(6): 889-891.
University Grants Commission, Delhi for providing [15] Khanna, N. And Bhatia, J., 2003, Action of Ocimum
financial support (UGC/NET-JRF Fellowship) to carry sanctum (Tulsi) in mice: possible mechanism
out the work. involved. J Ethnopharmacology, 88(2–3): 293–296.
[16] Kim OK, Murakami A, Nakamura Y, Ohighashi H.
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